Gamma-aminobutyric acid type A receptors (GABAARs) mediate the majority of synaptic inhibition in the brain and the actions of drugs such as benzodiazepines, barbiturates and anesthetics. Recently, mutations in the receptor have been linked to epilepsy. The long-term goal of our research program is to understand the function of the GABAAR in terms of its molecular structure. Work during the current project period significantly advanced our understanding of the structure of the GABA and benzodiazepine (BZD) binding sites. Experiments proposed herein build on this information to advance our understanding, on a structural level, of how GABA binding triggers channel activation and how BZD binding is coupled to receptor modulation. We propose to 1) identify local movements within the GABA binding site that couple binding to gating, 2) identify residues in the juxta-pore region that couple movements in the binding site to movements in the transmembrane domains and 3) identify residues that directly couple the GABA and BZD binding sites. The approach combines site-directed mutagenesis, disulfide crosslinking, mutant cycle analysis, substituted cysteine accessibility method, patch-clamping and kinetic analysis. The successful completion of these aims will not only increase our understanding of how GABAARs function in health and disease states but will also establish testable hypotheses for elucidating how other related ligand-gated ion channels function.